This invention relates to the treatment of mammalian sperm to modulate sperm function.
Although morphologically complete and capable of vigorous motility when they leave the male reproductive tract, mammalian sperm are not immediately able to fertilize oocytes. The acquisition of the capacity to fertilize an oocyte is termed capacitation. Once capacitated, sperm can undergo the acrosome reaction, a prerequisite for penetration of the zona pellucida and fusion with the oocyte plasma membrane; however sperm that have undergone a spontaneous acrosome reaction are non-fertilizing. A defect in these mechanisms results in a condition of infertility. At present there is no real treatment for this problem. The present invention is based on the discovery that the hormone calcitonin acts upon mammalian sperm to stimulate fertility.
Throughout the following description reference is made to various publications in the literature; a full reference list of these is given at the end of this specification.
Calcitonin is a 32-amino acid hypocalcemic hormone whose chief function is the regulation of Ca2+ fluxes and metabolism. There are three main phylogenetic classes of calcitonin, teleost/avian, artiodactyl and rat/human (Pozvek et al, 1997.). Teleost/avian calcitonin is the most potent, with salmon calcitonin being widely used to treat human metabolic bone disorders. The term xe2x80x98calcitoninxe2x80x99 as used herein refers to calcitonin of any species including calcitonin of salmon, eel, chicken, porcine, bovine, rat and human origin, as well as precursors and derivatives which have similar activities in vivo and in vitro. Synthetic equivalents may also be used.
In the early 1980s several studies reported the presence of high concentrations of calcitonin in human seminal plasma. These findings prompted a number of preliminary studies addressing the possibility that calcitonin might have an effect on sperm, and one study reported that some concentrations of salmon calcitonin, but not human calcitonin, had a negative effect on human sperm motility. Since 1990 there has been no further published information on this subject.
Using a robust and well-characterized mouse in vitro capacitation system, we have determined that calcitonin elicits biologically important responses in mammalian sperm. For comparison, fertilization promoting peptide (FPP), which has been shown to stimulate capacitation and fertilizing ability in uncapacitated mouse sperm and then to inhibit spontaneous acrosome loss in capacitated cells, was used as a positive control. Cells incubated for a short time with/without calcitonin were assessed for changes in the chlortetracycline (CTC) fluorescence patterns that are known to reflect changes in sperm function. Because salmon calcitonin is considerably more bioactive than human calcitonin, we have used both forms but at different concentrations. Salmon calcitonin at 5 ng/ml and human calcitonin at 200 ng/ml and 20 ng/ml significantly stimulated capacitation, as did FPP, the positive control: there were more capacitated, acrosome-intact (B pattern) cells and correspondingly fewer uncapacitated, acrosome-intact (F pattern) cells. This change in distribution between the uncapacitated (non-fertilizing) and the capacitated (potentially fertilizing) cells in calcitonin-treated suspensions has also been shown to correlate with a significant stimulation of fertilizing ability. In addition the effects of salmon calcitonin on capacitated mouse sperm were evaluated, again using FPP as a positive control. Results indicated that salmon calcitonin, like FPP, significantly inhibited spontaneous acrosome loss, and that this response was abolished by the inclusion of pertussis toxin; the latter result indicates the possible involvement of G proteins. Taken together, these responses in mouse sperm indicate that calcitonin both stimulates the acquisition of fertilizing ability and then holds the sperm in a potentially fertilizing state.
The earlier study mentioned above reported that salmon, but not human, calcitonin when used at higher concentrations inhibited human sperm motility (Gnessi et al, 1984), but no such effect was seen on mouse sperm. In our hands, treated samples showed more vigorous sperm motility than untreated controls, consistent with the changes in CTC patterns observed. Our results provide convincing evidence that calcitonin elicits significant responses in mammalian sperm, indicative of its potential as an endogenous stimulator/regulator of sperm fertilizing ability in vivo. This finding provides a new therapeutic strategy for treating human male subfertility. It also has applications for improving the success of artificial insemination in agriculturally-important animal species.
Sperm Suspension Preparation for Chlortetracycline Analysis
The contents of cauda epididymides (3-4 cauda per ml of medium) from mature TO male mice (Harlan Olac, Bicester, U.K.) were released into 2 ml modified Tyrode""s medium (Fraser, 1993) in 30 mm sterile culture dishes and allowed to disperse for 5 min. For studies on uncapacitated sperm, suspensions were then filtered through short columns of Sephadex G-25 (medium grade; Pharmacia, Uppsala, Sweden) to remove non-motile cells. Filtered suspensions were aliquotted out to different treatment groups, treated with nothing (control), calcitonin or FPP (as a positive control) and then incubated for 40 min. Salmon calcitonin was used at a final concentration of 5 ng/ml, human calcitonin was used at 200 ng/ml and 20 ng/ml and FPP was used at 100 nM. These short-term incubations were carried out in 0.5 ml plastic microcentrifuge tubes at 37xc2x0 C. in an atmosphere of 5% CO2, 5% O2, 90% N2. After the time allowed for incubation, an aliquot of each treated suspension was analysed using CTC to determine whether any effects on capacitation and hence acquisition of fertilizing ability could be observed. Three replicate experiments using uncapacitated sperm were carried out using salmon calcitonin and the higher concentration of human calcitonin (n=3); for the lower concentration of the human hormone, n=2.
In all experiments, a drop of each suspension was examined briefly for subjective motility evaluation. None of the experimental treatments had a deleterious effect on motility; judged subjectively, they all promoted more vigorous motility than that observed in untreated control samples.
Chlortetracycline Fluorescence Analysis
Chlortetracycline (CTC) fluorescence analysis was carried out as described by Green et al. (1994). Assessments were made on an Olympus BX40 microscope equipped with phase contrast and BX-FLA epifluorescence optics using the wide blue-violet excitation cube (U-MWBV). The excitation beam passed through a 400-440 nm band pass filter and CTC fluorescence was observed through a DM 455 dichroic mirror. In each sample, at least 100 sperm were classified as expressing one of three patterns: F, with fluorescence over the entire head, a pattern characteristic of uncapacitated (non-fertilizing), acrosome-intact sperm; B, with a fluorescence-free band in the postacrosomal region, a pattern characteristic of capacitated (potentially fertilizing), acrosome-intact sperm; AR, with dull or absent fluorescence over the whole head, a pattern characteristic of acrosome-reacted (non-fertilizing) sperm.
Sperm Preparation for in vitro Fertilization
The contents of the cauda epididymides from 2 mature TO male mice were released into 2 ml modified Tyrode""s medium in 30 mm sterile culture dishes and allowed to disperse for 5 min. Suspensions were then divided into 3 treatment groups, each in a 30 mm sterile dishes, and treated with nothing (Control) or with calcitonin. As in the CTC experiments, salmon calcitonin was used at a final concentration of 5 ng/ml and human calcitonin at 200 ng/ml. Each suspension was overlaid with autoclaved liquid paraffin and incubated for 40 min at 37xc2x0 C. in an atmosphere of 5% O2, 5% CO2, 90% N2.
In vitro Fertilization Analysis
Mature female TO mice were induced to superovulate by intraperitoneal injections of 7.5 IU equine chorionic gonadotrophin and approximately 50 hr later with 5 IU human chorionic gonadotrophin (hCG). Approximately 15 hr post-hCG, oviducts were removed and cumulus masses containing the oocytes were released into medium covered with liquid paraffin. Preincubated sperm suspensions were diluted xcx9c10 fold into medium of the same composition used for initial incubation (i.e., without or with calcitonin); 400 xcexcl of diluted suspension were transferred to culture dishes, covered with liquid paraffin and oocytes were added. After 65 min coincubation, oocytes were transferred to small droplets of control medium and then at 75 min fixed with buffered formalin (4% formaldehyde in phosphate buffered saline).
Oocytes were stained with 0.75% aceto orcein, mounted and assessed (Fraser, 1993). They were considered to be fertilized if they had resumed the second meiotic division and contained a decondensing sperm head (n=4).
Results